A novel approach of spectrophotometric quantification of chitosan based on one-step depolymerization with sodium nitrite followed by reaction of the end product with thiobarbituric acid has been proposed, optimized, and validated. In this process, chitosan is converted into 2,5-anhydro-D-mannose that reacts with thiobarbituric acid to form pink color. The color that resulted from the reaction was stabilized and measured at 555？nm. The method optimization was essential as many procedural parameters influenced the accuracy of the determination including hydrolysis conditions, thiobarbituric acid concentration, reaction time, pH, reaction temperature, and color stability period. Under given optimized conditions that appeared to be critical, chitosan was quantitatively analyzed and the calibration graph was linear over the range of 10–50？μg/mL ( ). This approach was applied for determination of chitosan in pharmaceutical formulation (chitocal) and had a recovery rate of higher than 96%. The developed method is easy to use and highly accurate. 1. Introduction Recently, much attention has been paid to chitosan as a potential polysaccharide resource. Chitosan is a linear amino polysaccharide of glucosamine and N-acetylglucosamine units and is obtained by alkaline deacetylation of chitin [1]. The following major characteristics of chitosan make this polymer advantageous for numerous applications: (1) it has a defined chemical structure; (2) it can be chemically and enzymatically modified; (3) it is physically and biologically functional; (4) it is biodegradable and biocompatible with many organs, tissues, and cells; (5) it can be processed into several products including flakes, fine powders, beads, membranes, sponges, cottons, fibers, and gels. Therefore, chitosan becomes of great interest as a new functional material of high potential in various fields and the discovery or development of methods for chitosan determination is imperative [2–5]. Although research activities dealing with chitosan are numerous, a generally accepted simple method for direct quantitative analysis is lacking. Chitosan can be degraded to glucosamine monomer by hydrolysis, and there are several reports on the determination of glucosamine by chromatographic, colorimetric, and fluorimetric techniques, or a combination of these [6–8]. Eikenes et al. [6] developed a method for determination of chitosan in wood and water samples based on acidic hydrolysis of chitosan to glucosamine followed by online derivatization by o-phthalaldehyde, chromatographic separation, and fluorescent detection.
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